Cyclopamine analogs

ABSTRACT

The invention provides derivatives of cyclopamine having the following formula:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/936,919, filed Mar. 27, 2018, now allowed, which is a continuation ofU.S. application Ser. No. 15/289,887, filed Oct. 10, 2016, now U.S. Pat.No. 9,951,083, issued Apr. 24, 2018, which is a continuation of U.S.application Ser. No. 14/203,297, filed Mar. 10, 2014, now U.S. Pat. No.9,492,435, issued Nov. 15, 2016, which is a continuation of U.S.application Ser. No. 12/199,722, filed Aug. 27, 2008, now U.S. Pat. No.8,669,365, issued Mar. 11, 2014, which is a continuation of U.S.application Ser. No. 11/965,688, filed Dec. 27, 2007, now U.S. Pat. No.7,812,164, issued Oct. 12, 2010, which claims benefit under 35 U.S.C. §119(e) to U.S. Application No. 60/941,596, filed Jun. 1, 2007 and U.S.Application No. 60/878,018, filed Dec. 28, 2006. The contents of theseapplications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention generally relates to cyclopamine analogs andpharmaceutical compositions thereof, and methods for preparing suchanalogs and compositions. These compounds and compositions are usefulfor the treatment of disorders mediated by the hedgehog pathway, such ascancer.

Inhibition of the hedgehog pathway in certain cancers has been shown toresult in inhibition of tumor growth. For example, anti-hedgehogantibodies have been shown to antagonize the function of the hedgehogpathway and inhibit the growth of tumors. Small molecule inhibition ofhedgehog pathway activity has also been shown to result in cell death ina number of cancer types.

Research in this area has focused primarily on the elucidation ofhedgehog pathway biology and the discovery of new hedgehog pathwayinhibitors. Although inhibitors of the hedgehog pathway have beenidentified, there still exists the need to identify more potentinhibitors of the hedgehog pathway.

PCT publication WO 2006/026430 published 9 Mar. 2006 and assigned to thesame assignee as the present application, discloses a wide variety ofcyclopamine analogs, focusing on those with unsaturation in the A or Bring. In the present application, the surprisingly potent analogscontain completely saturated A and B rings.

SUMMARY OF THE INVENTION

The present invention relates to analogs of steroidal alkaloids, such ascyclopamine, pharmaceutical compositions containing these compounds, andmethods for preparing these compounds. In one embodiment, the presentinvention relates to a compound represented by the following structure:

or a pharmaceutically acceptable salt thereof;

wherein R¹ is H, alkyl, —OR, amino, sulfonamido, sulfamido, —OC(O)R⁵,—N(R⁵)C(O)R⁵, or a sugar;

R² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, nitrile, orheterocycloalkyl;

or R¹ and R² taken together form ═O, ═S, ═N(OR), ═N(R), ═N(NR₂), ═C(R)₂;

R³ is H, alkyl, alkenyl, or alkynyl;

R⁴ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR⁵, —C(O)R⁵, —CO₂R⁵,—SO₂R⁵, —C(O)N(R⁵)(R⁵), —[C(R)₂]_(q)—R⁵, —[(W)—N(R)C(O)]_(q)R⁵,—[(W)—C(O)]_(q)R⁵, —[(W)—C(O)O]_(q)R⁵, —[(W)—OC(O)]_(q)R⁵,—[(W)—SO₂]_(q)R⁵, —[(W)—N(R⁵)SO₂]_(q)R⁵, —[(W)—C(O)N(R⁵)]_(q)R⁵,—[(W)—O]_(q)R⁵, —[(W)—N(R)]_(q)R⁵, —W—NR⁵ ₃ ⁺X⁻ or —[(W)—S]_(q)R⁵;

wherein each W is independently a diradical;

each q is independently 1, 2, 3, 4, 5, or 6;

X⁻ is a halide;

each R⁵ is independently H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or —[C(R)₂]_(p)—R⁶;wherein p is 0-6; or any two occurrences of R⁵ can be taken together toform a 4-8 membered optionally substituted ring which contains 0-3heteroatoms selected from N, O, S, and P;

each R⁶ is independently

hydroxyl, —N(R)COR, —N(R)C(O)OR, —N(R)SO₂(R), —C(O)N(R)₂, —OC(O)N(R)(R),—SO₂N(R)(R), —N(R)(R), —COOR, —C(O)N(OH)(R), —OS(O)₂OR, —S(O)₂OR,—OP(O)(OR)(OR), —NP(O)(OR)(OR), or —P(O)(OR)(OR); and

-   -   each R is independently H, alkyl, alkenyl, alkynyl, aryl,        cycloalkyl or aralkyl;    -   provided that when R², R³, and R⁴ are H; R¹ is not hydroxyl or a        sugar; further provided that when R⁴ is hydroxyl, then R¹ is not        a sugar or hydroxyl, and R¹ and R² are not C═O.

In certain embodiments, R¹ is H, hydroxyl, alkoxyl, aryloxy, or amino.In other embodiments, R¹ and R² taken together along with the carbon towhich they are bonded, form ═O, ═N(OR), or ═S. In other embodiments, R³is H and/or R⁴ is H, alkyl, hydroxyl, aralkyl, —[C(R)₂]_(q)—R⁵,—[(W)—N(R)C(O)]_(q)R⁵, —[(W)—N(R)SO₂]_(q)R⁵, —[(W)—C(O)N(R)]_(q)R⁵,—[(W)—O]_(q)R⁵, —[(W)—C(O)]_(q)R⁵, or —[(W)—C(O)O]_(q)R⁵. In otherembodiments, R¹ is H or —OR, R² is H or alkyl, and R⁴ is H. In others,R² is H or alkyl, R³ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, or aralkyl; and/or R⁴ is H, alkyl, aralkyl,—[(W)—N(R)C(O)]_(q)R⁵, —[(W)—N(R)SO₂]_(q)R⁵, —[(W)—C(O)N(R)]_(q)R⁵,—[(W)—O]_(q)R⁵, —[(W)—C(O)]_(q)R⁵, or —[(W)—C(O)O]_(q)R⁵. In otherembodiments, R¹ is sulfonamido.

In another embodiment, the present invention relates to a compoundselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compounds mentioned above are isolated.

In another embodiment, the present invention relates to an isolatedcompound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to a compoundrepresented by the following structure:

or a pharmaceutically acceptable salt thereof;

wherein R¹ is H, alkyl, —OR, amino, sulfonamido, sulfamido, —OC(O)R⁵,—N(R⁵)C(O)R⁵, or a sugar;

R² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, nitrile, orheterocycloalkyl;

or R¹ and R² taken together form ═O, ═S, ═N(OR), ═N(R), ═N(NR₂), ═C(R)₂;

R³ is H, alkyl, alkenyl, or alkynyl;

R⁴ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR⁵, —C(O)R⁵, —CO₂R⁵,—SO₂R⁵, —C(O)N(R⁵)(R⁵), —[C(R)₂]_(q)—R⁵, —[(W)—N(R)C(O)]_(q)R⁵,—[(W)—C(O)]_(q)R⁵, —[(W)—C(O)O]_(q)R⁵, —[(W)—OC(O)]_(q)R⁵,—[(W)—SO₂]_(q)R⁵, —[(W)—N(R⁵)SO₂]_(q)R⁵, —[(W)—C(O)N(R⁵)]_(q)R⁵,—[(W)—O]_(q)R⁵, —[(W)—N(R)]_(q)R⁵, —W—NR⁵ ₃ ⁺X⁻, or —[(W)—S]_(q)R⁵;

wherein each W is, independently, a diradical;

each q is, independently, 1, 2, 3, 4, 5, or 6;

X⁻ is a halide;

each R⁵ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or —[C(R)₂]_(p)—R⁶;wherein p is 0-6; or any two occurrences of R⁵ can be taken together toform a 4-8 membered optionally substituted ring which contains 0-3heteroatoms selected from N, O, S, and P;

each R⁶ is, independently, hydroxyl, —N(R)COR, —N(R)C(O)OR, —N(R)SO₂(R),—C(O)N(R)₂, —OC(O)N(R)(R), —SO₂N(R)(R), —N(R)(R), —COOR, —C(O)N(OH)(R),—OS(O)₂OR, —S(O)₂OR, —OP(O) (OR)(OR), —NP(O)(OR)(OR), or —P(O)(OR)(OR);

each R is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkylor aralkyl;

each of R⁷ and R^(7′) is H; or R⁷ and R^(7′) taken together form ═O;

R⁸ and R⁹ are H; or

R⁸ and R⁹ taken together form a bond; and

provided that when R³, R⁴, R⁸, R⁹ are H and R⁷ and R^(7′) taken togetherform ═O; R¹ cannot be hydroxyl and R² cannot be H;

provided that when R³, R⁴, R⁸, R⁹ are H and, R⁷ and R^(7′) takentogether form ═O; R¹ cannot be acetate and R² cannot be H;

provided that when R³, R⁴, R⁸, R⁹ are H and, R⁷ and R^(7′) are H; R¹ andR² taken together cannot be ═O; and

provided that when R³, R⁴, R⁸, R⁹ are H and, R⁷ and R^(7′) are H; R¹ andR² cannot be H.

In some embodiments, the compound is epimerically pure and/or isolated.In other embodiments, R⁴ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR⁵,—[C(R)₂]_(q)—R⁵, —[(W)—N(R)C(O)]_(q)R⁵, —[(W)—C(O)]_(q)R⁵,—[(W)—C(O)O]_(q)R⁵, —[(W)—OC(O)]_(q)R⁵, —[(W)—SO₂]_(q)R⁵,—[(W)—N(R⁵)SO₂]_(q)R⁵, —[(W)—C(O)N(R⁵)]_(q)R⁵, —[(W)—O]_(q) ⁵,—[(W)—N(R)]_(q)R⁵, or —[(W)—S]_(q)R⁵. Each of R⁷ and R^(7′) can be H. Inaddition, R¹ and R² taken together form ═O.

In another embodiment, the present invention relates to a compoundselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the above compounds are epimerically pure and/orisolated.

In another embodiment, the present invention relates to an epimericallypure compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a pharmaceuticalcomposition including any of the aforementioned compounds, and apharmaceutically acceptable excipient.

In one embodiment, the present invention relates to a process forpreparing cyclopropyl derivatives of cyclopamine and related analogshaving the formula 136:

wherein

Y is CR⁷R⁸;

R¹ is H, alkyl, amino, hydroxyl, carboxyl, carbamoyl, alkoxy, hydroxyl,sugar or a protected hydroxyl group;

R² is H, alkyl, alkenyl, alkynyl, nitrile, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or R¹ and R²taken together form ═O, ═S, ═N(OR⁹), ═N(R⁹), ═C(R⁹)₂, or ═N(N(R⁹)₂);

each of R³, R⁴, and R⁵ is, independently, H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, orheteroaralkyl; or R³ and R⁴ or R⁴ and R⁵ taken together form a bond;

R⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR⁹, —C(O)R⁹, —CO₂R⁹,—SO₂R⁹, —C(O)N(R⁹)(R⁹), —[C(R⁹)₂]_(q)R⁹, —[(W)—N(R⁹)C(O)]_(q)R⁹,—[(W)—C(O)]_(q)R⁹, —[(W)—C(O)O]_(q)R⁹, —[(W)—OC(O)]_(q)R⁹,—[(W)—SO₂]_(q)R⁹, —[(W)—N(R⁹)SO₂]_(q)R⁹, —[(W)—C(O)N(R⁹)]_(q)R⁹,—[(W)—O]_(q)R⁹, —[(W)—N(R⁹)]_(q)R⁹, —[(W)—S]_(q)R⁹, or a nitrogenprotecting group; wherein each W is independently a diradical; each q isindependently 1, 2, 3, 4, 5, or 6;

each of R⁷ and R⁸ is, independently, H, alkyl, alkenyl, aryl, nitrile,amido, halide, or ester; and

each R⁹ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl.

The process includes the steps of contacting a compound of formula 136awith a haloalkylzinc phosphate cyclopropanating agent to yield acompound of formula 136:

wherein

R¹, R², R³, R⁴, R⁵, R⁶ are as defined in compound 136.

In another embodiment, the present invention provides methods forpreparing a compound of formula 137:

wherein

Y is CR⁷R⁸;

R¹ is H, alkyl, amino, hydroxyl, carboxyl, carbamoyl, alkoxy, hydroxyl,sugar or a protected hydroxyl group;

R² is H, alkyl, alkenyl, alkynyl, nitrile, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or R¹ and R²taken together form ═O, ═S, ═N(OR⁹), ═N(R⁹), ═C(R⁹)₂, or ═N(N(R⁹)₂);

each of R³, R⁴, and R⁵ is, independently, H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, orheteroaralkyl; or R³ and R⁴ or R⁴ and R⁵ taken together form a bond;

R⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR⁹, —C(O)R⁹, —CO₂R⁹,—SO₂R⁹, —C(O)N(R⁹)(R⁹), —[C(R⁹)₂]_(q)R⁹, —[(W)—N(R⁹)C(O)]_(q)R⁹,—[(W)—C(O)]_(q)R⁹, —[(W)—C(O)O]_(q)R⁹, —[(W)—OC(O)]_(q)R⁹,—[(W)—SO₂]_(q)R⁹, —[(W)—N(R⁹)SO₂]_(q)R⁹, —[(W)—C(O)N(R⁹)]_(q)R⁹,—[(W)—O]_(q)R⁹, —[(W)—N(R⁹)]_(q)R⁹, —[(W)—S]_(q)R⁹, or a nitrogenprotecting group;

wherein each W is, independently, a diradical;

each q is independently 1, 2, 3, 4, 5, or 6;

each of R⁷ and R⁸ is, independently, H, alkyl, alkenyl, aryl, nitrile,amido, halide, or ester; and

each R⁹ is independently H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl. The processincludes the steps of: first contacting a compound of formula 137a witha haloalkylzinc phosphate cyclopropanating agent;

wherein

R¹, R², R³, R⁴, R⁵, R⁶ are as defined in compound 137; to form acompound with formula 137b:

wherein

R¹, R², R³, R⁴, R⁵, R⁶ and Y are as defined in compound 137; and thencontacting the compound of formula 137b with an acid to give a compoundof formula 137.

In certain embodiments, R⁷ and R⁸ can both be H; in other embodiments R¹can be a protected hydroxyl; and/or R⁶ is a nitrogen protecting group.

In certain embodiments, the haloalkylzinc phosphate cyclopropanatingagent is formed by combining a phosphoric acid of formula 141a, adialkylzinc, and a dihaloalkylane of formula 141b:

wherein

each of X and X′ is, independently, chloride, bromide, or iodide;

each of R⁷ and R⁸ is, independently, H, alkyl, halide, amido, nitro, orester;

each of R¹⁰ and R¹¹ is, independently, alkyl, alkenyl, aralkyl, aryl,heteroaryl, heteroaralkyl;

or R¹⁰ and R¹¹ taken together have the formula 141c, 141 d, or 141e;

wherein

m is, independently for each occurrence, 0, 1, 2, 3, or 4; n is,independently for each occurrence, 0, 1, or 2; and each of R¹², R¹³,R¹⁴, R¹⁵, R¹⁶, R¹⁷ and R¹⁸ is, independently, alkyl, aryl, aralkyl, orhalide.

In another embodiment, the present invention relates to a process forpreparing a compound of formula 142:

The process includes the steps of contacting a compound of formula 142awith a cyclopropanating agent to form a compound formula 142b; and

combining the compound of formula 142b with an acid to give the compoundof formula 142;

wherein

Y is CR⁷R⁸; R¹ is a protected hydroxyl group;

R² is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, or heteroaralkyl; each of R³, R⁴, and R⁵ is,independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or

R³ and R⁴ or R⁴ and R⁵ taken together form a bond; R⁶ is a nitrogenprotecting group;

each of R⁷ and R⁸ is, independently, H, alkyl, alkenyl, aryl, nitrile,amido, halide, or ester; and

each R⁹ is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl.

In certain embodiments, R⁷ and R⁸ can both be H; in other embodimentsthe protected hydroxyl group can be an ester or a carbonate; thenitrogen protecting can be a carbamate or an amide; R⁷ and R⁸ can bothbe H and the nitrogen protecting can be a carbamate or an amide; R² andR³ can be H and R⁴ and R⁵ taken together can form a bond; and/or thecyclopropanating agent is generated from a dihaloalkane and a metalspecies (e.g., dialkyl zinc or a zinc copper couple).

In certain embodiments the cyclopropanating agent is generated from adihaloalkane species and a dialkyl zinc species, and a phosphoric acidspecies or a salt thereof. The phosphoric acid species can have astructure of formula 151:

or a salt thereof;

wherein

each of R¹⁰ and R¹¹ is independently alkyl, alkenyl, aralkyl, aryl,heteroaryl, heteroaralkyl; or R¹⁰ and R¹¹ taken together have theformula 151a, 151b, or 151c;

wherein

m independently for each occurrence is 0, 1, 2, 3, or 4; n independentlyfor each occurrence is 0, 1, or 2; each of R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷and R¹⁸ is, independently, alkyl, aryl, aralkyl, or halide.

In certain embodiments the acid is a Bronsted acid (e.g., acetic acid,trifluoromethanesulfonic acid, phosphoric acid, methanesulfonic acid orHCl). In other embodiments the acid is a Lewis acid (e.g., BF₃, zincchloride, zinc methanesulfonate, or a zinc salt).

The present invention also relates to a process for preparing a compoundof formula 156:

The process includes the steps of:

contacting a compound of formula 156a with a cyclopropanating agent toform a compound formula 156b; and

combining the compound of formula 156b with an acid to give the compoundof formula 156; where R¹ is an oxygen protecting group selected from thegroup consisting of formate, acetate, chloroacetate, dichloroacetate,trichloroacetate, pivaloate, benzoate, alkyl carbonate, alkenylcarbonate, aryl carbonates, aralkyl carbonate, 2,2,2-trichloroethylcarbonate, alkoxymethyl ether, aralkoxymethyl ether, alkylthiomethlether, aralkylthio ether, arylthio ether, trialkylsilyl ether,alkylarylsilyl ether, benzyl ether, arylmethyl ether, allyl ether; andR² is a nitrogen protecting group selected from the group consisting offormyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, phenyl acetyl,benzoyls, alkyl carbamates, aralkyl carbamates, aryl carbamates, allyl,aralkyl, triarylmethyl, alkoxymethyl, aralkoxymethyl, N-2-cyanoethyl,diarylphosphinamides, dialkylphosphinamidates, diarylphosphinamidates,and trialkylsilyl.

In certain embodiments the cyclopropanating agent is formed by combininga phosphoric acid of formula 58a, a dialkylzinc, and a dihaloalkylane offormula 158b:

wherein

each of X and X′ is, independently, chloride, bromide, or iodide; eachof R⁷ and R⁸ is, independently, H, alkyl, halide, amido, or ester; eachof R¹⁰ and R¹¹ is, independently, alkyl, alkenyl, aralkyl, aryl,heteroaryl, heteroaralkyl; or R¹⁰ and R¹¹ taken together have theformula 158c, 158 d, or 158e;

wherein

m independently for each occurrence is 0, 1, 2, 3, or 4; n independentlyfor each occurrence is 0, 1, or 2; each of R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷and R¹⁸ is, independently, alkyl, aryl, aralkyl, or halide.

The oxygen protecting group can be, in some embodiments, selected fromalkyl carbonates, aralkyl carbonates (e.g., benzylcarbonate), benzoates,pivaloate, or formate. The nitrogen protecting group can be selectedfrom benzoyl, trichloroacetyl, trifluoroacetyl, formyl, alkylcarbamates, aralkyl carbamates (e.g., benzylcarbamate), aryl carbamates,diarylphosphinamides, dialkylphosphinamidates, ordiarylphosphinamidates.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The definitions of terms used herein are meant to incorporate thepresent state-of-the-art definitions recognized for each term in thechemical and pharmaceutical fields. Where appropriate, exemplificationis provided. The definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The term “acylamino” refers to a moiety that may be represented by thegeneral formula:

wherein R50 and R54 independently represent a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R61,

where R61 represents an aryl, a cycloalkyl, a cycloalkenyl, aheterocycle or a polycycle; and m is zero or an integer in the range of1 to 8.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” refers to an alkyl group, as definedabove, having an oxygen radical attached thereto. Representative alkoxylgroups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has 30 or fewer carbon atoms in itsbackbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain),20 or fewer. Likewise, certain cycloalkyls have from 3-10 carbon atomsin their ring structure, and others have 5, 6 or 7 carbons in the ringstructure.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R61, R61 represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8, or R50 and R51, taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure. Certain embodiments of the amide in thepresent invention will not include imides which may be unstable.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50 and R51 (and optionally R52) each independently represent ahydrogen, an alkyl, an alkenyl, or —(CH₂)_(m)—R61, where R61 is definedas above. Thus, the term “alkylamine” includes an amine group, asdefined above, having a substituted or unsubstituted alkyl attachedthereto, i.e., at least one of R50 and R51 is an alkyl group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, anthracene, naphthalene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term “Bronsted acid” refers to any substance that can act as ahydrogen ion (proton) donor.

The term “carboxyl” is defined to include esters, thiocarboxyl,aldehydes, ketones and the like and thus includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and each ofR55 and R56 represents independently a hydrogen, an alkyl, an alkenyl,—(CH₂)_(m)—R61, where m and R61 are defined above.

The term “diradical” refers to any of a series of divalent groups fromalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl groups. For example,

is an alkyl diradical;

is also an alkyl diradical;

is an aralkyl diradical; and

is an (alkyl)heteroaralkyl diradical. Typical examples include alkylenesof general structure (CH₂)_(x) where X is 1-6, and correspondingalkenylene and alkynylene linkers having 2-6 carbon atoms and one ormore double or triple bonds; cycloalkylene groups having 3-8 ringmembers; and aralkyl groups wherein one open valence is on the aryl ringand one is on the alkyl portion such as

and its isomers.

The term “haloalkyl”, as used herein, refers to an alkyl group whereanywhere from 1 to all hydrogens have been replaced with a halide. A“perhaloalkyl” is where all of the hydrogens have been replaced with ahalide.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Examples of heteroatoms include boron,nitrogen, oxygen, phosphorus, sulfur and selenium.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, in some instances from 3- to 7-memberedrings, whose ring structures include one to four heteroatoms.Heterocycles can also be polycycles. Heterocyclyl groups include, forexample, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones, and the like. Theheterocyclic ring may be substituted at one or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “isolated” in connection with a compound of the presentinvention means the compound is not in a cell or organism and thecompound is separated from some or all of the components that typicallyaccompany it in nature.

The term “Lewis acid” refers to any substance that can act as anelectron pair acceptor.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, in some embodiments from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Certain alkyl groups are lower alkyls. In someembodiments, a substituent designated herein as alkyl is a lower alkyl.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The term “oxo” refers to a carbonyl oxygen (═O).

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle may be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “epimerically pure” in connection with a compound of thepresent invention means that the compound is substantially free ofstereoisomers of the compound wherein the configuration of thestereogenic center that R³ is bonded to is inverted. For example anepimerically pure compound represented by the following formula:

wherein R¹, R², R³, R⁴, R⁷, R^(7′), R⁸, and R⁹ are as defined below, issubstantially free of compounds represented by the following formula:

wherein R¹, R², R³, R⁴, R⁷, R^(7′), R⁸, and R⁹ are as defined below.Epimerically pure compounds contain less than about 20% by mass, lessthan about 15% by mass, less than about 10% by mass, less than about 5%by mass, or less than about 3% by mass of stereoisomeric compoundswherein the configuration of the stereogenic center that R³ is bonded tois inverted relative to the compound.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991). In some cases, the functional group being protected and theprotecting group are together referred to as one moiety. For example,the fragment shown below is sometimes referred to as a benzyl carbonate;i.e., the protected (underlined) O makes up part of the carbonate.

Similarly, the fragment shown below, in which the protected N makes uppart of the carbamate, is referred to as a benzyl carbamate.

The term “sugar” as used herein refers to a natural or an unnaturalmonosaccharide, disaccharide or oligosaccharide comprising one or morepyranose or furanose rings. The sugar may be covalently bonded to thesteroidal alkaloid of the present invention through an ether linkage orthrough an alkyl linkage. In certain embodiments the saccharide moietymay be covalently bonded to a steroidal alkaloid of the presentinvention at an anomeric center of a saccharide ring. Sugars mayinclude, but are not limited to ribose, arabinose, xylose, lyxose,allose, altrose, glucose, mannose, gulose, idose, galactose, talose,glucose, and trehalose.

The term “sulfonamido” or “sulfonamide” as used herein includes a moietyhaving either of the following formulae:

wherein R50 and R56 are as defined above.

The terms “triflyl”, “tosyl”, “mesyl”, and “nonaflyl” refer totrifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, andnonafluorobutanesulfonyl groups, respectively. The terms “triflate”,“tosylate”, “mesylate”, and “nonaflate” to trifluoromethanesulfonateester, p-toluenesulfonate ester, methanesulfonate ester, andnonafluorobutanesulfonate ester functional groups and molecules thatcontain the groups, respectively.

The term “thioxo” refers to a carbonyl sulfur (═S).

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the compounds of the presentinvention include the conventional nontoxic salts or quaternary ammoniumsalts of the compounds, e.g., from non-toxic organic or inorganic acids.For example, such conventional nontoxic salts include those derived frominorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Synthesis of Steroidal Alkaloid Compounds

The ring expanded steroidal alkaloid derivatives described above can beprepared directly from naturally occurring steroidal alkaloids orsynthetic analogs thereof. In certain instances, the steroidal alkaloidstarting materials can be cyclopamine or jervine. These steroidalalkaloids can be purchased commercially or extracted from Veratrumcalifornicum. Briefly, the process of the present invention comprisesthe steps of cyclopropanating suitable starting steroidal alkaloidderivatives followed by ring expansion rearrangement of the cyclopropylderivatives. In some instances, it may be desirable to suitably protector otherwise transform reactive functionalities present on the moleculeprior to cyclopropanation. For example, an alcohol present at R¹ and asecondary nitrogen present on the fused furano-piperidine ring can bothbe protected prior to cyclopropanation. In certain embodiments,protecting groups that are efficiently added and removed from thealkaloid, yield intermediates in the synthetic process with improvedhandling properties and which allow for the efficient purification ofthe synthetic intermediates formed may be preferred.

Examples of oxygen protecting groups include, but are not limited toformate, acetate, chloroacetate, dichloroacetate, trichloroacetate,pivaloate, benzoates, alkyl carbonate, alkenyl carbonate, arylcarbonates, aralkyl carbonate (e.g., benzyl carbonate),2,2,2-trichloroethyl carbonate, alkoxymethyl ether, aralkoxymethylether, alkylthiomethl ether, aralkylthio ether, arylthio ether,trialkylsilyl ether, alkylarylsilyl ether, benzyl ether, arylmethylether, and allyl ether.

Examples of nitrogen protecting groups include, but are not limited toformyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, phenyl acetyl,benzoyls, benzamides, alkyl carbamates, aralkyl carbamates (e.g., benzylcarbamates), aryl carbamates, allyl, aralkyl, alkoxymethyl,aralkoxymethyl, N-2-cyanoethyl, diarylphosphinamides,dialkylphosphinamidates, diarylphosphinamidates, and trialkylsilyl.

Additional protecting groups that may be used in the methods of thepresent invention are described in Green T. W.; Wuts P. G. ProtectiveGroups in Organic Synthesis 3^(rd) Edition, John Wiley & Sons, Inc.1999.

A variety of cyclopropanating agents can be used to cyclopropanate thesteroidal alkaloid. 1,1-haloalkylmetal complexes and reactive speciesreferred to as carbenoids, are commonly used to cyclopropanate olefins.These reagents are typically made using a diiodoalkane or diazoalkaneand a metal or organometalic species such as Et₂Zn, iBu₃Al, samarium,copper, rhodium, or palladium. In certain embodiments, Et₂Zn anddiiodomethane are used to generate the 1,1-haloalkylmetal species.

The reactivity and the ease of handling of the 1,1-haloalkylzinccomplexes can be modified by the addition of certain reagents, such asacids. It is believed that the addition of an acid to the1,1-haloalkylzinc species generates an alkyl zinc mixed salt. In theexamples described below a biarylphosphoric acid is combined withdiiodomethane and diethylzinc to generate a putative haloalkyl zincphosphate cyclopropanating agent. A variety of phosphoric acids can beused to generate the putative haloalkylzinc phosphate.

Other known cyclopropanation methods such as those utilizing sulfurylides to react with an olefin conjugated to a carbonyl to add a CH2 orCH-alkyl or CH-aryl group, and metal-catalyzed decomposition ofdiazoalkyl and α-diazo-carbonyl compounds, such as diazomethane andethyl diazoacetate, can also be used: these methods readily providecyclopropanes having alkyl, aryl, alkoxycarbonyl (—COOR), or acylsubstituents. Additional cyclopropanating agents are described inMasalov et al., Organic Letters (2004) Vol. 6, pp. 2365-8 and Hansen etal., Chem. Comm. (2006) 4838-40.

The cyclopropyl ring may be substituted or unsubstituted. In cases wherethe cyclopropyl ring is substituted, the groups attached to themethylene of the cyclopropane will be installed onto the D ring afterrearrangement and ring expansion.

The cyclopropanation reactions may be conducted in an aprotic solvent.Suitable solvents include ethers, such as diethyl ether,1,2-dimethoxyethane, diglyme, t-butyl methyl ether, tetrahydrofuran andthe like; halogenated solvents, such as chloroform, dichloromethane,dichloroethane, and the like; aliphatic or aromatic hydrocarbonsolvents, such as benzene, xylene, toluene, hexane, pentane and thelike; esters and ketones, such as ethyl acetate, acetone, and2-butanone; polar aprotic solvents, such as acetonitrile,dimethylsulfoxide, dimethylformamide, and the like; or combinations oftwo or more solvents. In a certain embodiments, dichloromethane is thesolvent used for the cyclopropanation when a dialkyl zinc anddiiodomethane is used.

In the examples described below, a solution containing thecyclopropanating agent is prepared by first adding a solution of aphosphoric acid to a solution of diethylzinc, followed by addition ofdiiodomethane to the reaction solution. The cyclopropanation substrateis then added to this solution. Alternatively, the cyclopropanationagent can be prepared in the presence of the cyclopropanation substrateby changing the order of addition of the reagents. In certainembodiments, the cyclopropanation reaction is conducted by first addingthe phosphoric acid to a solution of dialkylzinc, followed by theaddition of the cyclopropanation substrate, and finally the dihaloalkaneis added. Using this method the cyclopropanating agent is generatedunder controlled conditions and immediately reacts with thecyclopropanation substrate. The cyclopropanation methods describedherein can also be used to cyclopropanate other polycyclic compounds,for example, those with steroidal backbones.

Following synthesis of the cyclopropanated steroidal alkaloid core, thecompound may be derivatized using a variety of functionalizationreactions known in the art. Representative examples include palladiumcoupling reactions to alkenylhalides or aryl halides, oxidations,reductions, reactions with nucleophiles, reactions with electrophiles,pericyclic reactions, radical reactions, installation of protectinggroups, removal of protecting groups, and the like.

In the presence of Lewis or Bronsted acids the cyclopropyl analogsundergo a rearrangement and ring expansion to afford steroidal alkaloidanalogs in which the D ring has been expanded by one carbon.

The cyclopropanation and ring expansion can take place in a two-step onereaction vessel process or in a two-step two reaction vessel process.When the cyclopropanation and ring expansion are conducted in the samereaction vessel the acid used to initiate the ring expansionrearrangement is added after completion of the cyclopropanationreaction. Under certain conditions, the zinc salts that are generated inthe course of cyclopropanating the steroidal alkaloid can themselves actas Lewis acids to catalyze the ring expansion rearrangement. Thereactivity of the zinc salts generated after the cyclopropanation can bemodified by the addition of acids to generate more active Lewis acids.

As described below in the examples section, the methanesulfonic acid isadded to the cyclopropanation reaction vessel after completion of thecyclopropanation. Additional examples of suitable acids include, but arenot limited to zinc salts, boron compounds, magnesium salts, titaniumsalts, indium salts, aluminum salts, tin salts, lanthanum salts,trifluoromethanesulfonic acid, diaryloxyphosporic acids, acetic acid,and HCl. In a certain embodiments of the invention the Lewis acid usedis a zinc salt or BF₃.

These ring expanded analogs may be further functionalized using avariety of functionalization reactions known in the art. Representativeexamples include palladium coupling reactions to alkenylhalides or arylhalides, oxidations, reductions, reactions with nucleophiles, reactionswith electrophiles, pericyclic reactions, radical reactions,installation of protecting groups, removal of protecting groups, and thelike.

Utility of Compounds

Hedgehog signaling is essential in many stages of development,especially in formation of left-right symmetry. Loss or reduction ofhedgehog signaling leads to multiple developmental deficits andmalformations, one of the most striking of which is cyclopia.

Many tumors and proliferative conditions have been shown to depend onthe hedgehog pathway. The growth of such cells and survival can beaffected by treatment with the compounds of the present invention.Recently, it has been reported that activating hedgehog pathwaymutations occur in sporadic basal cell carcinoma (Xie et al. (1998)Nature 391: 90-2) and primitive neuroectodermal tumors of the centralnervous system (Reifenberger et al. (1998) Cancer Res 58: 1798-803).Uncontrolled activation of the hedgehog pathway has also been shown innumerous cancer types such as GI tract cancers including pancreatic,esophageal, gastric cancer (Berman et al. (2003) Nature 425: 846-51,Thayer et al. (2003) Nature 425: 851-56) lung cancer (Watkins et al.(2003) Nature 422: 313-317, prostate cancer (Karhadkar et al (2004)Nature 431: 707-12, Sheng et al. (2004) Molecular Cancer 3: 29-42, Fanet al. (2004) Endocrinology 145: 3961-70), breast cancer (Kubo et al.(2004) Cancer Research 64: 6071-74, Lewis et al. (2004) Journal ofMammary Gland Biology and Neoplasia 2: 165-181) and hepatocellularcancer (Sicklick et al. (2005) ASCO conference, Mohini et al. (2005)AACR conference).

For example, small molecule inhibition of the hedgehog pathway has beenshown to inhibit the growth of basal cell carcinoma (Williams, et al.,2003 PNAS 100: 4616-21), medulloblastoma (Berman et al., 2002 Science297: 1559-61), pancreatic cancer (Berman et al., 2003 Nature 425:846-51), gastrointestinal cancers (Berman et al., 2003 Nature 425:846-51, published PCT application WO 05/013800), esophageal cancer(Berman et al., 2003 Nature 425: 846-51), lung cancer (Watkins et al.,2003. Nature 422: 313-7), and prostate cancer (Karhadkar et al., 2004.Nature 431: 707-12).

In addition, it has been shown that many cancer types have uncontrolledactivation of the hedgehog pathway, for example, breast cancer (Kubo etal., 2004. Cancer Research 64: 6071-4), heptacellular cancer (Patil etal., 2005. 96^(th) Annual AACR conference, abstract #2942 Sicklick etal., 2005. ASCO annual meeting, abstract #9610), hematologicalmalignancies (Watkins and Matsui, unpublished results), basal carcinoma(Bale & Yu, 2001. Human Molec. Genet. 10:757-762 Xie et al., 1998 Nature391: 90-92), medulloblastoma (Pietsch et al., 1997. Cancer Res. 57:2085-88), and gastric cancer (Ma et al., 2005 Carcinogenesis May 19,2005 (Epub)). As shown in the Examples, the compounds disclosed hereinhave been shown to modulate the hedgehog pathway, and selected compoundshave been shown to inhibit tumor growth. It is therefore believed thatthese compounds can be useful to treat a variety of conditions, such asvarious cancers.

Pharmaceutical Compositions

In another embodiment, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. The pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for buccal,sublingual, and systemic absorption, capsules, boluses, powders,granules, pastes for application to the tongue; (2) parenteraladministration, for example, by subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension,or sustained-release formulation; (3) topical application, for example,as a cream, ointment, or a controlled-release patch or spray applied tothe skin; (4) intravaginally or intrarectally, for example, as apessary, cream or foam; (5) sublingually; (6) ocularly; (7)transdermally; (8) pulmonarily, or (9) nasally.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, dispersing agents, lubricants,and/or antioxidants. Prevention of the action of microorganisms upon thecompounds of the present invention may be ensured by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, about 0.1 to 99%, orabout 10 to 50%, or about 10 to 40%, or about 10 to 30, or about 10 to20%, or about 10 to 15% of active ingredient in combination with apharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenousand subcutaneous doses of the compounds of the present invention for apatient, when used for the indicated effects, will range from about0.0001 to about 100 mg, or about 0.001 to about 100 mg, or about 0.01 toabout 100 mg, or about 0.1 to about 100 mg per, or about 1 to about 50mg per kilogram of body weight per day.

The subject receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1

Step A

Recrystallized cyclopamine 2 (14.1 g, 34.0 mmol, 1 eq) is dissolved inanhydrous DCM (70 mL) and anhydrous MeOH (29 mL). The clear solution iscooled, and triethylamine (10.4 g, 102.7 mmol, 3 eq) followed by benzylchloroformate (6.20 g, 36.3 mmol, 1.1 eq) is added. After the additionis complete, the solution is stirred in the ice bath for 30 min. Threeportions of benzyl chloroformate (3×0.35 g, 3.46 mmol, 0.03 eq) areadded over the 3 h. The reaction is slowly quenched with water (71 mL),while maintaining the temperature below 20° C. The mixture is stirredfor 15 min before the layers are settled and separated. The organiclayer is dried over sodium sulfate and filtered. The combined filtrateis buffered with anhydrous pyridine (30 mL), concentrated, and solventexchanged with additional anhydrous pyridine (43 mL) and concentrated.

The solution of the compound in pyridine (43 mL) is further diluted withadditional anhydrous pyridine (85 mL). Trimethylacetyl chloride (8.3 g,68.7 mmol, 2 eq) is added slowly to the reaction mixture, and thereaction is heated to 45° C. The reaction is stirred at 45° C. for 30min. The reaction is cooled and quenched by the addition of anhydrousMeOH (4.5 mL). The quenched reaction mixture is stirred at rt for 40 minand then diluted with toluene (97 mL) and is treated sequentially withwater (35 mL) and a 10 wt % aqueous sodium carbonate solution (100 mL).After vigorous stirring, the layers are separated and the organic layeris washed twice with water (2×100 mL), dried over sodium sulfate, andfiltered. The filter cake is rinsed with toluene (49 mL) and discarded.The combined filtrates are concentrated, and solvent exchanged withconcentration to toluene (145 mL) and further concentrating to dryness.The product is recrystallized from toluene and heptane. The crystallineproduct is isolated by suction filtration, washed with cold heptane anddried to a constant weight to afford 15.1 g of the desired product.

Step B

Bis(2,6-dimethyphenyl)phosphate (10.65 g, 34.8 mmol, 3.1 eq) is dried byconcentration from anhydrous DCM (42 mL) and held under a nitrogenatmosphere. The phosphate is then redissolved in anhydrous DCM (110 mL).In a separate flask, a solution of neat diethylzinc (4.17 g, 33.8 mmol,3.0 eq) in anhydrous DCM (35 mL) is prepared and cooled to −25° C. Thephosphate solution is slowly transferred to the vessel containing thediethylzinc solution over 1 h, maintaining the temperature at or below−10° C. The clear ethylzinc phosphate solution is warmed to 0° C. andstirred for 15 min. Diiodomethane (9.25 g, 34.5 mmoles, 3.0 eq) isslowly added to the ethylzinc phosphate solution, maintaining thereaction temperature between 0 and 5° C. After the addition is complete,the zinc carbenoid solution is stirred for an additional 20 min.

In a separate flask, compound 3 (7.20 g, 11.4 mmol, 1 eq) is dissolvedin anhydrous DCM (36 mL) and transferred to the reaction flask. Afterthe addition is complete, the ice bath is removed and the reactionmixture is allowed to warm to rt. After 6 h the contents of the flaskare cooled to −53° C. A solution of methanesulfonic acid (3.38 g, 35.2mmol, 3.1 eq) in anhydrous DCM (3 mL) is added, maintaining the reactiontemperature below −45° C. After 10 min morpholine (20 g, 230 mmol, 20eq) is added to the reaction mixture, maintaining the reactiontemperature below −40° C. The reaction is allowed to warm to rtovernight. The morpholine salts are removed by filtration and the filtercake rinsed with DCM (22 mL). The combined filtrates are washed with 2Naqueous hydrochloric acid (2×140 mL), 5% aqueous sodium bicarbonate (140mL), 5% aqueous sodium bicarbonate (70 mL) and 5% aqueous sodiumbisulfite (70 mL), and brine (144 mL). The organic layer is dried overmagnesium sulfate and filtered. Without going to dryness, the DCMsolution is concentrated and solvent exchanged with methanol (280 mL).The suspension are chilled with an ice bath and stirred for 40 minutes.The solids are isolated by filtration, washed twice with cold methanol(2×25 mL), and dried to a constant weight to afford 5.94 g of thedesired product.

Step C

In a round bottom flask, compound 4 (11.67 g, 18.1 mmol, 1 eq) and 20%palladium hydroxide on wet carbon (2.40 g, 1.71 mmol, 0.09 eq) areplaced under a nitrogen atmosphere and diluted with EtOAc (115 mL) andtoluene (60 mL). The solution is degassed with nitrogen (3×) withevacuation/purge cycles, and the process is repeated for hydrogen. Thesuspension is vigorously stirred at rt for 1.5 h. The hydrogenatmosphere is replaced with nitrogen. Ethylenediamine (0.57 g, 9.5 mmol,0.52 eq) is added to the reaction, and the resulting mixture stirred for20 min. The solution is filtered under nitrogen, and the filtrate iswashed with a 2% (wt/wt) aqueous solution of ethylenediamine (125 mL)then water (130 mL), and then dried over sodium sulfate. The dryingagent is removed by filtration and the filtrate is concentrated todryness under vacuum. The solids that remained are chased with toluene(2×55 mL) on the rotary evaporator and the resulting material usedwithout further purification in the next step

The material from the previous step is dissolved in anhydrous DCM (26mL). The resulting clear solution is added to a 1 M solution of DIBAL inDCM (65 mL, 65 mmol, 3.6 eq) while maintaining the reaction temperaturebetween −10 and −25° C. After 30 min the reaction is quenched withacetone (13 mL), maintaining the reaction temperature at or below 0° C.After stirring the quenched reaction mixture for 17 min, it is added inportions to a flask containing a cold, stirred solution of 20% (wt/wt)aqueous Rochelle salt (200 mL). The resulting gelatinous suspension isstirred at rt for 15 h. After stirring, the clean layers are separatedand the aqueous layer back extracted with DCM (30 mL). The combinedorganic layers are washed with water (60 mL) and dried over sodiumsulfate. The drying agent is removed by filtration and discarded. Thefiltrate is concentrated under vacuum and solvent exchanged to toluene(225 mL added in portions). The resulting solution is furtherconcentrated to a suspension (50 mL) and diluted with heptane (115 mL).The resulting mixture is heated until turning homogeneous (92° C.). Thesolution is cooled slowly over 12 h to 15° C., and then held for 16additional h. The crystalline product is isolated by suction filtration,washed with heptane (2×75 mL) and dried to a constant weight to afford7.70 g of the desired product.

A round bottom flask is sequentially charged with the homo-allylicalcohol (7.50 g, 17.6 mmol, 1 eq), aluminum tri-tert-butoxide (6.10 g,24.8 mmol, 1.4 eq), anhydrous toluene (115 mL), and 2-butanone (90 g,1.24 mol, 7 eq). The suspension is heated under a nitrogen atmosphere to75° C. for 16 h. The reaction temperature is then allowed to cool to 49°C. Aqueous 20% (w/w) potassium sodium tartrate solution (226 g) is addedto the stirred suspension. The suspension is stirred at rt for 3.5 h.The layers are separated. The organic layer washed with aqueous 20%Rochelle salt (2×250 mL) and water (225 mL), then dried over sodiumsulfate and filtered. The residue is rinsed with toluene (30 mL) anddiscarded. The combined organics are concentrated to dryness. Residualreaction solvents are removed from the material by concentrating from2-propanol (250 mL added portion-wise) to a final solution mass of 44 g.Solvent exchange from 2-propanol to heptane (275 mL added portion-wise)to a final solution mass is 41 g fully precipitated the desired product.The suspension is diluted with of additional heptane (40 mL), stirred atrt for 1 h, and filtered. The product is washed with n-heptane (17 mL)and dried to afford 5.4 g of the desired product.

Step D

A round-bottom flask is charged with starting material (110 mg, 0.26mmol, 1 eq) and 10% palladium on carbon (106 mg). The solids aresuspended in pyridine (4 mL). The suspension is placed under hydrogenatmosphere (1 atm) and the mixture is stirred overnight at rt. Thereaction mixture is filtered through Celite® and the filtrateconcentrated in vacuo. The crude material is purified using silica gelflash chromatography (MeOH/DCM 5:95) to afford 93 mg of the desiredcompound. ([M+H]=426.6 m/z).

Example 2

Step A

Cyclopamine 2 (5.02 g, 12.2 mmol, 1.0 eq) is dissolved in anhydrouspyridine (25 mL). DMAP (300 mg, 2.44 mmol, 0.2 eq.) and triethylamine(5.5 mL, 39.1 mmol, 3.2 eq) are added, followed by BtO-Cbz (10.5 g, 39.1mmol, 3.2 eq) and heated at 40° C. for 2 h. The mixture is cooled to rt,treated with 30 mL water, heated to get a homogeneous solution andallowed to cool to room temp. The white precipitate that formed iscollected by filtration, the filter cake is washed with portions ofwater (3×50 mL), and dried in air to afford 9.53 g of crude materialwhich is crystallized from toluene/heptanes (1:9, 70 mL) to give 6.75 gof the desired product.

Step B

To a solution of diethyl zinc (572 mg, 482 μL, 4.63 mmol, 3.00 eq) in5.0 mL DCM at −20° C. is added a solution ofbis-(2,6-Dimethylphenyl)phosphoric acid (1.42 g, 4.63 mmol, 3.00 eq) inDCM (15 mL) maintaining the reaction temperature below −8° C. Thesolution is aged for 15 min. at 0° C., neat diiodomethane (1.24 g, 374μL, 3.00 eq) is added, aged for 15 min. at 0° C. before adding asolution of (BisCBzcyclopamine, 1.05 g, 1.54 mmol, 1.0 eq), in DCM (10mL). The cooling bath is replaced by a water bath at rt and maintainedat rt for 4.5 h. The mixture is cooled to −76° C. with a dry ice-acetonebath and treated drop wise with methanesulfonic acid DCM solution (0.6mL 50% v/v solution 4.63 mmol, 3.0 eq) maintaining the reactiontemperature below −74° C. The mixture is aged for 15-20 min. andquenched drop wise with morpholine (2.69 g, 2.70 mL, 20 eq) maintainingthe reaction temperature below −65° C. The cooling bath is removed, thereaction mixture is stirred for 16-18 h., the white precipitate isfiltered off, and the filtrate is successively washed with 2.0 M HCl(2×20 mL), satd. sodium bicarbonate (2×20 mL), water (2×20 mL) and brine(20 mL). Dried over magnesium sulfate, concentrated in vacuo to drynessand the crude is purified by silica gel flash chromatography(hexanes/EtOAc 17:3→4:1) to afford 924 mg (1.33 mmol, 86%) of thedesired product.

Step C

To a solution of compound 7 (4.05 g, 5.83 mmol, 1 eq) in a solution ofEtOAc:toluene (2:1, 60 mL) is added of 20% palladium hydroxide on carbon(823 mg, 0.583 mmol, 0.1 eq.). The flask is evacuated and filled withhydrogen three times. The mixture is stirred under an atmosphere ofhydrogen for 1 h. Neat ethylene diamine (0.38 mL) is added, stirred for1 h., and the catalyst is filtered off. The filter cake is washed twicewith EtOAc:toluene (2:1, 12 mL). The combined filtrates are washed witha 2% aqueous solution of ethylene diamine (3×20 mL), dried over sodiumsulfate and concentrated in vacuo to give 2.46 g as a white crystallinesolid.

Step D

A round bottom flask is sequentially charged with the homo-allylicalcohol 8 (7.50 g, 17.6 mmol, 1 eq), aluminum tri-tert-butoxide (6.10 g,24.8 mmol, 1.4 eq), anhydrous toluene (115 mL), and 2-butanone (90 g,1.24 mol, 7 eq). The suspension is heated under a nitrogen atmosphere to75° C. for 16 h. The reaction temperature is then allowed to cool to 49°C. Aqueous 20% (w/w) potassium sodium tartrate solution (226 g) is addedto the stirred suspension. The suspension is stirred at rt for 3.5 h.The layers are separated. The organic layer washed with aqueous 20%Rochelle's salt (2×250 mL) and water (225 mL), then dried over sodiumsulfate and filtered. The residue is rinsed with toluene (30 mL) anddiscarded. The combined organics are concentrated to dryness. Residualreaction solvents are removed from the material by concentrating from2-propanol (250 mL added portion-wise) to a final solution mass of 44 g.Solvent exchange from 2-propanol to n-heptane (275 mL addedportion-wise) to a final solution mass of 41 g fully precipitated thedesired product. The suspension is diluted with of additional n-heptane(40 mL), stirred at rt for 1 h, and filtered. The product is washed withn-heptane (17 mL) and dried to afford 5.4 g of the desired product.

Step E

A round-bottom flask is charged with starting material (110 mg, 0.26mmol, 1 eq) and 10% palladium on carbon (106 mg). The solids aresuspended in pyridine (4 mL). The suspension is placed under hydrogenatmosphere (1 atm) and the mixture is stirred overnight at rt. Thereaction mixture is filtered through Celite® and the filtrateconcentrated in vacuo. The crude material is purified using silica gelflash chromatography (MeOH/DCM 5:95) to afford 93 mg of the desiredcompound. ([M+H]=426.6 m/z).

Example 3

In a seal tube, ketone 6 (85 mg, 0.199 mmol, 1 equiv.) was charged andtriethyleneglycol (2 mL) was added followed by hydrazine monohydrate(500 mg, 10 mmol, 50 equiv.) and potassium carbonate (138 mg, 1 mmol, 5equiv.). The tube was sealed and the reaction was heated at 150° C. for16 h. The reaction was cooled to rt and water was added. The residue wasextracted with chloroform (3×). The combined organic layers are washedwith water, dried over Na₂SO₄, and concentrated to dryness. Thecolorless oil was purified using silica gel flash chromatography(DCM/MeOH 96:4). The purified fractions are pooled and concentrated todryness. The resulting oil was dissolved in MTBE and washed with water(2×), 2N NaOH, and then brine. The combined organic layers are driedover Na₂SO₄, filtered and evaporated to afford 64 mg of the desiredmaterial as a white foam. ([M+H]=412.7 m/z).

Example 4

A sealed tube was charged with compound 5 (223 mg, 0.52 mmol, 1 eq) andDMF (1 mL). 2-bromopropane (1.3 g, 10.5 mmol, 20 eq) and Na₂CO₃ (73 mg,0.68 mmol, 1.3 eq) were added and the flask was sealed and heated to 50°C. The mixture was stirred for 16 h at which point ˜70% conversion wasobserved. Additional (0.26 g, 2.12 mmol, 4 eq) was added. The reactionwas stirred for 2 h and additional 2-bromopropane (0.13 g, 1.1 mmol, 2eq) was added. The reaction was stirred for another 1 h. The reactionwas cooled to rt and water was added. The residue was extracted withMTBE (3×). The organic layers were combined washed with brine, driedover Na₂SO₄, filtered, and concentrated to dryness. The white foam waspurified using silica gel flash chromatography (DCM/MeOH 99:1) to give206 mg of the N-isopropyl derivative as a white foam.

The N-isopropyl derivative (205 mg, 0.44 mmol, 1 eq) was dissolved in of4-methoxypyridine (1.5 mL). The flask was placed under inert atmosphereand Pd/C 10% (wet, Aldrich Degussa type E101, 40 mg) was added. Theflask was sealed and purged three times with hydrogen and left 16 hunder 1 atm of hydrogen. Celite® was added to the reaction mixture. Themixture was filtered through a small pad of Celite® and washed withEtOAc. The organic layer was washed with 1N HCl aq. (2×) then withwater. The organic layer was dried over Na₂SO₄, filtered through cottonand evaporated to give 34 mg of crude. The aqueous layer was neutralizedwith 2N KOH and extracted with DCM (3×). The combined organic layerswere washed with water, dried over Na₂SO₄, filtered through cotton andcombined with the initial 34 mg of crude. The crude material waspurified using silica gel flash chromatography hexane/EtOAc (6:4) toafford 80 mg of desired product. ([M+H]=468.7 m/z).

Example 5

In a round-bottom flask, compound 6 (88 mg, 0.21 mmol, 1 eq) wasdissolved in anhydrous THF (1 mL). The mixture was cooled to 0° C.Pyridine (84 μL, 1 mmol, 5 eq) and benzoylperoxide (150 mg, 0.62 mmol, 3eq) were added successively. The homogeneous mixture was graduallywarmed to rt over 2 h and stirred overnight at rt. The reaction wasquenched by adding saturated NaHCO₃. The residue was extracted withMTBE. The combined organic layers were washed with water, dried overNa₂SO₄, filtered and concentrated under reduced pressure. The crude waspurified using silica gel flash chromatography (hexane/EtOAc (9:1 to4:1)) to give the N—O derivative product (60 mg, 0.11 mmol) as a whitefoam. This foam was dissolved in 2 mL of MeOH followed by 2N aqueous KOH(0.4 mL). The reaction mixture was stirred for 1 h. Most of the MeOH wasevaporated under a stream of nitrogen and 1N HCl (500 μL) was added. Thematerial was extracted with DCM (3×). The combined organic layers werewashed with water, dried over Na₂SO₄, filtered and concentrated underreduced pressure. The crude was purified using silica gel flashchromatography (hexanes/EtOAc (from 88:12→1:1)) to yield 11 mg of thedesired product. ([M+H]=442.5 m/z).

Example 6

Step A

In a round bottom flask, compound 6 (89 mg, 0.209 mmol, 1 eq) andN-(benzyloxy carbonyl)-aminoacetaldehyde (148 mg, 0.85 mmol, 4 eq) weredissolved in DCM (2 mL). Sodium triacetoxyborohydride (177 mg, 0.85mmol, 4 eq) was added and the reaction was stirred for 3 h at rt. Themixture was poured in saturated aqueous NaHCO₃ solution and the residuewas extracted with DCM (3×). The combined organic layers were washedwith water, dried over Na₂SO₄, filtered through cotton and evaporated togive a foamy solid (247 mg). The crude was dissolved in EtOAc (2 mL) andtreated with of 4 M HCl (156 μL). After 30 min a white precipitateslowly formed. The resulting slurry was stirred for 15 min. Filtrationgave 120 mg of white solid. The material was dissolved in EtOAc andtreated with a saturated aqueous NaHCO₃ solution. The organic layer wascollect and the aqueous layer and was extracted with EtOAc (2×). Thecombined organic layers were washed with brine, dried over Na₂SO₄.Filtration and evaporation gave the desired intermediate. This materialwas used in the next step without purification.

Step B

All of the material from Step A was dissolved in EtOAc (3 mL) andtreated with of Pd/C 10% (30 mg, wet, Aldrich Degussa type E101). Theflask was sealed and purged three times with hydrogen and left overnightunder 1 atm of hydrogen. After 16 h, the mixture was filtered through asmall pad of Celite® and washed with EtOAc to afford 52 mg of the amineas a white foam.

Step C

A round-bottom flask containing the amine 14 (52 mg, 0.11 mmol, 1 eq)was charged with the 1H-tetrazole-5-acetic acid (21 mg, 0.166 mmol, 1.5eq), DCM (2 mL), EDCI (42 mg, 0.22 mmol, 2 eq) andN,N-diisopropylethylamine (57 mg, 0.44 mmol, 4 eq) The resulting yellowsolution was stirred at rt for 4 h. The reaction was quenched by theaddition of saturated aqueous NaHCO₃ solution and the residue wasextracted with DCM (3×). The combined organic layers were dried overNa₂SO₄, filtered through cotton and evaporated to give 62 mg ofoff-white solid. This material was purified using silica gel flashchromatography (MeOH/DCM 5:95→10:90) to afford 31 mg of the desiredproduct. ([M+H]=579.7 m/z).

Example 7

A round-bottom flask was charged with starting material (47 mg, 0.110mmol, 1 eq) and potassium carbonate (150 mg, 1.09 mmol, 10 eq). Thesolids were suspended in 2 mL of DCM. Iodomethane (14 μL, 0.22 mmol, 2eq) was added and the mixture was stirred for 2 at rt. TLC (DCM/MeOH95:5) indicate >90% completion. Iodomethane (14 μL, 0.22 mmol, 2 eq) wasadded to the reaction mixture, which was stirred for 2 h. The reactionmixture was added water. The phases were separated and the organics weredried and concentrated to dryness. The residue was purified using silicagel flash chromatography (DCM/MeOH 100:0→98:2) afford 34 mg of thedesired product ([M+H]=440.5 m/z).

Example 8

A round-bottom flask was charged with starting material (59 mg, 0.126mmol, 1 eq) and potassium carbonate (350 mg, 2.5 mmol, 20 eq). Thesolids were suspended in 3 mL of DCM. The reaction was charged withiodomethane (80 μL, 1.29 mmol, 10 eq) and the mixture was stirredovernight at rt. The reaction mixture was charged with water. Theorganic phase was separated and the aqueous layer was back extractedwith DCM. The combined organic layers were dried and concentrated todryness. The residue was purified using silica gel flash chromatography.DCM/MeOH (95:5→90:10) to afford 52 mg of the desired product.([M+H]=639.5 m/z).

Example 9

Step A

In a round bottom flask, compound 5 (50 mg, 0.12 mmol, 1 eq) andN-(t-butoxycarbonyl)-aminoacetaldehyde (6 mg, 0.38 mmol, 3.1 eq) weredissolved in DCM (2 mL). Sodium triacetoxyborohydride (8 mg, 0.38 mmol,3.1 eq) was added and the reaction was stirred for 2 h at rt. Themixture was poured in saturated aqueous NaHCO₃ solution and the residuewas extracted with DCM (3×). The combined organic layers were washedwith water, dried over Na₂SO₄, filtered through cotton and evaporated togive a foamy solid (95 mg). The crude material was purified using silicagel flash chromatography (EtOAc/Hexanes 1:1) to yield 55 mg of compound18.

Step B

A round-bottom flask was charged with starting material 18 (800 mg, 1.4mmol, 1 eq). The solid was dissolved in a solution of DCM and TFA (10mL, 1:1). The solution was stirred for 45 min at rt. The reaction waspartitioned between a solution of 10% sodium carbonate and DCM. Theorganic was separated and washed with 10% sodium carbonate. The organicphase was concentrated to dryness. The residue was used without furtherpurification for the next step.

Step C

A round-bottom flask was charged with starting material (300 mg, 0.64mmol, 1 eq) was dissolved in THF/ACN (1:1, 4 mL). The reaction wascharged 37% formaldehyde in water (240 μL, 3.22 mmol, 5 eq) and sodiumcyanoborohydride (64 mg, 1 mmol, 1.6 eq). The mixture was stirred for 30min at rt. The reaction was then partitioned between a solution asaturated aqueous solution of sodium bicarbonate and DCM. The organicwas separated, dried and concentrated to dryness. The crude material waspurified using silica gel flash chromatography (MeOH/DCM 5:95→10:90) togive the desired material.

Step D

A round-bottom flask was charged with starting material 20 (30 mg, 0.06mmol, 1 eq) and 10% palladium on carbon (30 mg). The solids weresuspended in pyridine (2 mL). The suspension was placed under hydrogenatmosphere and the mixture was stirred overnight at rt. The reactionmixture was filtered on Celite® and the filtrate concentrated todryness. The crude material was purified using silica gel flashchromatography (MeOH/DCM 5:95→10:90) to give the desired material.([M+H]=497.7 m/z).

Example 10

A round-bottom flask was charged with starting material (85 mg, 0.20mmol, 1 eq) was dissolved in DCM (4 mL). The reaction was charged withN-(2-oxoethyl)acetamide (80 mg, 0.70 mmol, 3.5 eq) and sodiumtriacetoxyborohydride (170 mg, 0.80, 4 eq). The mixture was stirred for1 hour at rt. The reaction was partitioned between a solution asaturated aqueous solution of sodium bicarbonate and DCM. The organicwas separated, dried and concentrated to dryness. The crude material waspurified using silica gel flash chromatography (MeOH/DCM 5:95) to givethe desired material. ([M+H]=511.7 m/z).

Example 11

Compound 22 was synthesized according to the procedure described inexample 9, using N-methyl-N-(2-oxoethyl)acetamide in place ofN-(2-oxoethyl)acetamide. ([M+H]=525.7 m/z).

Example 12

Compound 23 was synthesized according to the procedure described inexample 10, using N-(2-oxoethyl)-3-phenylpropanamide in place ofN-(2-oxoethyl)acetamide. ([M+H]=601.8 m/z).

Example 13

Compound 23 was synthesized according to the procedure described inexample 10, using N-methyl-N-(2-oxoethyl)-3-phenylpropanamide in placeof N-(2-oxoethyl)acetamide. ([M+H]615.9 m/z)

Example 14

Step A

A round-bottom flask was charged with compound 6 (4.23 g, 9.94 mmol, 1eq) and THF (60 mL). Triethylamine (6.92 mL, 49.7 mmol, 5.0 eq) andbenzyl chloroformate (1.54 mL, 10.93 mmol, 1.1 eq) were added and themixture was stirred for 1 hour at rt. The reaction mixture waspartitioned between saturated aqueous bicarbonate (100 mL) and EtOAc(100 mL). The phases were separated and the organics were dried (Na₂SO₄)and concentrated to dryness. The crude material was purified usingsilica gel flash chromatography (EtOAc/Hexanes 2:98→14:86) to give 3.75g of material.

Step B

A MeOH solution (10 ml) of cerium trichloride heptahydrate (260 mg, 0.69mmol, 1.3 eq.) at 0° C. was treated with sodium borohydride (24 mg, 0.65mmol, 1.2 eq), stirred for 15 min, and then cooled to −78° C. A THFsolution (10 ml) of ketone 26 (300 mg, 0.54 mmol, 1 eq) was added, andthe mixture was stirred for 1 h and then warmed to rt. Water (50 ml) andEtOAc (50 ml) were added, mixed, and the layers split. The organic layerwas collected, washed with brine (30 ml), dried over sodium sulfate, andconcentrated to a white residue. The crude product was purified bysilica gel flash chromatography (ether/hexanes 2:3→1:1) to give 235 mgof 3-beta alcohol 27.

Step C

Compound 27 (235 mg, 0.42 mmol, 1 eq) was dissolved in EtOAc (7 ml) in aflask with stir bar and rubber septum. The solution was sparged withnitrogen, and Pd/C 10% (wet, Aldrich Degussa type E101, 50 mg) wasadded. This mixture was sparged with nitrogen and then hydrogen gas andstirred at rt for 3 h. The mixture was then sparged with nitrogen,filtered through a 0.45 μm polyethylene membrane and concentrated to aclear oil. The oil was purified by silica gel flash chromatography(NH₄OH(aq)/MeOH/DCM 0.5:2:97.5→0.5:6:93.5) to give 130 mg of compound 25as a white powder. ([M+H]=427.4 m/z)

Example 15

Step A

A THF solution (10 ml) of ketone 26 (300 mg, 0.54 mmol, 1 eq) at −78° C.was treated with K-Selectride® (Potassium tri-sec-butylborohydride)(0.58 ml, 0.58 mmol, 1.1 eq) and stirred for 60 min. Methanol (1 ml) wasadded and the solution warmed to rt. Water (50 ml) and EtOAc (50 ml)were added, mixed, and the layers split. The organic layer was washedwith brine (30 ml), dried over sodium sulfate, and concentrated to awhite residue. The crude product was purified by silica gel flashchromatography (Ether/Hexanes 2:3→1:14) to give 170 mg of pure 3-alphaalcohol 29.

Step B

Compound 29 (170 mg, 0.30 mmol, 1 eq) was dissolved in EtOAc (5 ml) in aflask with stir bar and rubber septum. The solution was sparged withnitrogen, and Pd/C 10% (wet, Aldrich Degussa type E101, 35 mg) wasadded. This mixture was sparged with nitrogen and then hydrogen gas andstirred at rt for 3 h. The mixture was then sparged with nitrogen,filtered through a 0.45 μm polyethylene membrane and concentrated to aclear oil. The oil was purified by silica gel flash chromatography(NH₄OH(aq)/MeOH/DCM 0.5:2:97.5→0.5:6:93.5) to afford 76 mg of compound28 as a white powder ([M+H]=427.4 m/z).

Example 16

Step A

Compound 27 (100 mg, 0.18 mmol, 1 eq) with benzyltriethylammoniumchloride (8 mg, 0.36 mmol, 0.2 eq) was dissolved in DCM (5 ml) andstirred vigorously with dimethyl sulfate (130 μL, 1.43 mmol, 8 eq) and50% aqueous potassium hydroxide (0.5 ml) at rt for 18 h. The mixture waspartitioned between water (30 ml) and EtOAc (30 ml), and the organiclayer was then washed with brine, dried over sodium sulfate, andconcentrated to a clear oil. The crude ether was purified by silica gelflash chromatography (Ether/Hexanes 3:7→9:113) to give 75 mg of themethyl ether as a clear oil.

Step B

Compound 31 (66 mg, 0.115 mmol, 1 eq) was dissolved in EtOAc (5 ml) in aflask with stir bar and rubber septum. The solution was sparged withnitrogen, and Pd/C 10% (wet, Aldrich Degussa type E101, 20 mg) wasadded. This mixture was sparged with nitrogen and then hydrogen gas andstirred at rt for 3 h. The mixture was then sparged with nitrogen,filtered through a 0.45 μm polyethylene membrane and concentrated to aclear oil. The oil was purified by silica gel flash chromatography(NH₄OH(aq)/MeOH/DCM 0.5:2:97.5→0.5:6:93.5) to give 22 mg of compound 30as a white powder ([M+H]=441.4 m/z).

Example 17

Step A

Compound 27 (100 mg, 0.18 mmol, 1 eq) was dissolved in DCM (5 ml), and4-dimethylaminopyridine (4 mg, 0.35 mmol, 0.2 eq),N,N-diisopropylethylamine (0.15 ml, 0.9 mmol, 5 eq), and aceticanhydride (0.070 ml, 0.72 mmol, 4 eq) were added. After stirring for 12h at rt, the solution was split between EtOAc (30 ml) and 5% aqueoussodium bicarbonate (15 ml). The organic layer was washed with brine,dried over sodium sulfate, and concentrated to a clear oil. The crudeester was purified by silica gel chromatography (Ether/Hexanes3:7→9:113) to give 100 mg of the ester as a clear oil.

Step B

Compound 33 (100 mg, 0.18 mmol, 1 eq) was dissolved in EtOAc (5 ml) in aflask with stir bar and rubber septum. The solution was sparged withnitrogen, and Pd/C 10% (wet, Aldrich Degussa type E101, 20 mg) wasadded. This mixture was sparged with nitrogen and then hydrogen gas andstirred at rt for 3 h. The mixture was then sparged with nitrogen,filtered through a 0.45 μm polyethylene membrane and concentrated to aclear oil. The oil was purified by silica gel flash chromatography(NH₄OH(aq)/MeOH/DCM 0.5:2:97.5→0.5:6:93.5) to give 45 mg of compound 32as a white powder ([M+H]=469.4 m/z).

Example 18

Compound 34 was synthesized according to the procedure described inexample 16, using compound 29 in place of compound 27. ([M+H]=441.4m/z).

Example 19

Compound 34 was synthesized according to the procedure described inexample 17, using compound 29 in place of compound 27. MS ([M+H]=469.4m/z)

Example 20

Step A

An ethanol solution (5 ml) of compound 26 (185 mg, 0.3 mmol, 1 eq) wastreated with hydroxylamine hydrochloride (140 mg, 2 mmol, 6 eq), sodiumacetate (160 mg, 2 mmol, 6 eq), and water (0.5 mL), and the mixture wasstirred at rt for 1 hr. The mixture was split between EtOAc and water(50 mL each). The organic layer was washed with brine (30 mL), driedover sodium sulfate, and concentrated to a white residue. The crudeproduct was purified by silica gel chromatography (ether/hexanes2:3→1:1) to give 193 mg of oxime 37.

Step B

Compound 37 (65 mg, 0.113 mmol) was dissolved in EtOAc (7 ml) in a flaskwith stir bar and rubber septum. The solution was sparged with nitrogen,and Pd/C 10% (wet, Aldrich Degussa type E101, 20 mg) was added. Thismixture was sparged with nitrogen and then hydrogen gas and stirred atrt for 3 h. The mixture was then sparged with nitrogen, filtered througha 0.45 μm polyethylene membrane and concentrated to a clear oil. The oilwas purified by silica gel flash chromatography (NH₄OH(aq)/MeOH/DCM0.5:2:97.5→0.5:6:93.5) to give 15 mg of compound 36 as a white powder, amixture of cis and trans oxime isomers ([M+H]=440.3 m/z).

Example 21

Step A

Compound 27 (42 mg, 0.075 mmol, 1 eq) was dissolved in DCM (5 ml), and4-dimethylaminopyridine (2 mg, 0.02 mmol, 0.2 eq), N-Cbz glycine (23 mg,0.110 mmol, 1.5 eq), and diisopropylcarbodiimide (0.023 ml, 0.150 mmol,2 eq) were added. After stirring for 12 h at rt, the solution was splitbetween EtOAc (30 ml) and 5% aqueous sodium bicarbonate (15 ml). Theorganic layer was washed with brine, dried over sodium sulfate, andconcentrated to a clear oil. The crude ester was purified by silica gelflash chromatography (ether/hexanes 2:3→1:1) to give 35 mg of the esteras a clear oil

Step B

Compound 39 (235 mg, 0.42 mmol, 1 eq) was dissolved in EtOAc (7 mL) in aflask with stir bar and rubber septum. The solution was sparged withnitrogen, and Pd/C 10% (wet, Aldrich Degussa type E101, 50 mg) wasadded. This mixture was sparged with nitrogen and then hydrogen gas andstirred at rt for 3 h. The mixture was then sparged with nitrogen,filtered through a 0.45 μm polyethylene membrane and concentrated to aclear oil. The oil was purified by silica gel flash chromatography(NH₄OH(aq)/MeOH/DCM 0.5:2:97.5→0.5:6:93.5) to give 17 mg of the desireproduct as a white powder ([M+H]=452.4 m/z).

Example 22

Compound 40 was synthesized according to the procedure described inexample 21, using compound 29 in place of compound 27. ([M+H]=452.4 m/z)

Example 23

Compound 41 was synthesized according to the procedure described inexample 10, using N-(2-oxoethyl)-2-phenylacetamide in place ofN-(2-oxoethyl)acetamide. ([M+H]=587.7 m/z).

Example 24

Step A

A round-bottom flask was charged with alcohol 29 (7.60 g, 13.53 mmol, 1eq) and was dissolved in DCM (115 mL). The reaction was charged withtriethylamine (8.21 g, 81 mmol, 6.0 eq). The mixture was cooled to 0° C.and charged with methanesulfonylchloride (1.86 g, 16.2 mmol, 1.2 eq).After 30 min, the reaction mixture was partitioned between a saturatedaqueous solution of sodium bicarbonate and EtOAc. The organic layer wasseparated, dried over sodium sulfate and concentrated to dryness. Theresidue was purified using silica gel flash chromatography(EtOAc/hexanes 10→25%) gave the desired material mesylate.

A round-bottom flask was charged with the mesylate (9.1 g, 14.22 mmol, 1eq) and was dissolved in 50 mL of DMPU. The reaction was charged withsodium azide (4.62 g, 71.1 mmol, 5.0 eq) and heated to 60° C. Themixture was stirred for 17 h. The reaction mixture was then cooled to rtand charged with water. The mixture was stirred for 30 min. The mixturewas filtered under vacuum, rinsed with water and air dried and useddirectly without purification in the next step.

Step B

A round-bottom flask was charged with azide 43 (8.35 g, 14.23 mmol, 1eq) and THF (120 mL) was added. The reaction was then charged withtriphenylphosphine (11.2 g, 42.7 mmol, 3.0 eq). The mixture was heatedto 50° C. and stirred for 20 h. The reaction mixture was then cooled tort and the solvent removed under vacuum. The residue purified usingsilica gel flash chromatography (MeOH/DCM 10%→20%) to afford the amine.

A round-bottom flask was charged with the amine (5.10 g, 9.09 mmol, 1eq) and was dissolved in DCM (60 mL). The reaction was charged withN,N-diisopropylethylamine (5.88 g, 45.5 mmol, 5.0 eq). The mixture wascooled to 0° C. and charged with methanesulfonylchloride (2.08 g, 18.2mmol, 2.0 eq). After 30 minutes, the reaction mixture was partitionedbetween a saturated aqueous solution of sodium bicarbonate and EtOAc.The organic layer was collected, dried over sodium sulfate andconcentrated to dryness. The residue was purified using silica gel flashchromatography (EtOAc/hexanes 10→30%) to afford the Cbz protectedmethanesulfonamide.

Step C

A round-bottom flask was charged with the Cbz protectedmethanesulfonamide (5.37 g, 8.41 mmol, 1 eq) and 10% palladium on carbon(1.0 g). The solids were suspended in 2-propanol (50 mL). The suspensionwas placed under hydrogen atmosphere and the mixture was stirred for 4 hat 25° C. The reaction mixture was then filtered on Celite® and thefiltrate concentrated to dryness. The residue was then purified usingsilica gel flash chromatography (DCM/MeOH 0→5%) to afford the desiredproduct. [M+H]=505.6 m/z.

Alternate Synthesis of Compound 42

Recrystallized cyclopamine (2.07 g) is charged to an appropriately sizedreaction vessel and placed under an inert atmosphere. EtOAc (7.6 g),triethylamine (1.53 g), and DMAP (307 mg) are added sequentially. Thesuspension is warmed to 40° C. Cbz-OBt is added in three portions over90 minutes, keeping the internal temperature below 45° C. The reactionmixture is stirred at 40° C. for 90 minutes. The temperature ismaintained while methanol (26.4 g) is slowly added to the reactionmixture. The resulting suspension is cooled to room temperature andstirred for at least 15 hours. The crude product is collected byfiltration and rinsed with methanol (5 g). The white solid is driedunder vacuum to a constant weight and recrystallized from heptane (30.3g) and toluene (3.2 g) to afford Compound 24a (3.0 g).

Solid bis(2,6-dimethylphenyl) hydrogenphosphate and 24a are pre-driedand placed under a nitrogen atmosphere. Neat diethyl zinc (722 mg) ischarged to an appropriately sized reaction vessel containing DCM (9.0g). DCM solutions of the phosphate (1.83 g in 17.9 g) and IPI-332690(1.34 g in 3.6 g) are added sequentially at or below 25° C.Diiodomethane (1.58 g) is charged and the reaction is stirred at 28° C.for 4-6 hours. The reaction is cooled to −45° C. and a solution ofmethanesulfonic acid in DCM (566 mg in 1.5 g) is charged. After 15minutes, morpholine (1.711 g) is added and the mixture is allowed towarm to room temperature overnight. The organic layer is washed twicewith 2N HCl (2×13.6 g) then sequentially with 4.8 wt % sodium carbonate(aq), 4.8 wt % sodium sulfite (aq), and 4.8 wt % brine (13.6 g each).The organic layer is dried, filtered, concentrated to 4 g and dilutedwith isopropanol (4 g). The product is crystallized from solution by theslow addition of methanol (9.3 g). Filtration with a methanol rinse (2.6g) and drying afford 1.09 g of 24b (79% isolated yield).

Johnson Matthey Pd/C catalyst A-305038-5 (890 mg) is charged to anappropriately sized reaction vessel, followed by 24b (2.24 g). Thereaction vessel is purged with N₂ and toluene (21.8 g) and 2-propanol(6.7 g) are added sequentially. The system is degassed and placed undera nitrogen atmosphere, and the process is repeated with hydrogen. Thesystem is stirred vigorously and the hydrogen blanket is maintained atone atmosphere for 4-5 hours. The reaction is monitor by either TLC orHPLC. If incomplete, the reaction is inerted, additional catalyst (145mg) is charged, and the hydrogen atmosphere is returned for anotherhour. Ethylenediamine (12.9 mg) is charged and the mixture was stirredfor 15 minutes. The catalyst is removed by filtration with a toluene:IPA(3:1) rinse. The filtrate and rinses are concentrated and solventexchanged to toluene. The product is crystallized from toluene (19.0 g)and heptane (18.0 g) to afford 24c as a white crystalline solid (1.34 g,98% yield).

24c (644 mg) is charged to an appropriately sized reaction vesselfollowed by aluminum t-butoxide (525 mg), toluene (8.34 g, 15 vol), and2-butanone (7.83 g, 15 vol). The contents of the flask are degassed withevacuation/nitrogen purge cycles to remove oxygen and the reactionmixture is heated at 75° C. with vigorous stirring for 16-18 hours. Thereaction is quenched by the addition of aqueous Rochelle's salt (2.6 gin 10.3 g water) and the mixture vigorously stirred for one hour at 45°C. The aqueous and organic layers are separated. The aqueous layer isback extracted with a mixture of toluene (2.9 g) and EtOAc (2.9 g). Theorganic layers are combined and washed with fresh Rochelle's saltsolution (2.6 g in 10.3 g water) and then with water (12.9 g). Theresulting organic layer is dried over sodium sulfate (1.97 g), filtered,and concentrated in vacuo. The product is crystallized via a charge andconcentration solvent exchange first to IPA (6.5 g) and then Heptane(7.7 g). The thick heptane slurry (˜2.7 g) is stirred overnight andsolids are collected by filtration. Vacuum drying affords 24 d (550 mg)in an 85% yield.

The enone 24 d (459 mg) and Johnson-Matthey 5% palladium on carbon(A503023-5, 101 mg) are charged to an appropriately sized multi neckreaction vessel. The vessel is purged with nitrogen and 3-picoline (2.2g) is charged as the solvent. Stirring is started and the vessel isfirst degassed using nitrogen and then stirred under hydrogen atatmospheric pressure for 8 hours. At the end of the reaction, thecatalyst is removed by filtration through 0.2 micron media, rinsing withACN (1.4 ml). The filtrate and rinse are combined in a clean reactionvessel equipped with mechanical stirring, an internal temperature probe,and a nitrogen atmosphere. A solution of citric acid (3.7 g) in water(9.2 ml) is charged to the reaction vessel at or below 30° C., andIPI-335589 is allowed to slowly crystallize from solution as the citratesalt at 20 and then 0° C. The crystalline product is recovered bysuction filtration and washed with water (3.7 ml). After drying, thecitrate salt, 24e, is isolated as a hydrate (3-5 wt % water) in 89.5%yield (622 mg) with a β:α ratio approaching 90:1.

24e (1.50 g) is charged to the appropriately sized reactor along with2-methyltetrahydrofuran (7.7 g) and 1M sodium carbonate (9.0 ml) Asolution of benzyl chloroformate (454 mg) in 2-methyltetrahydrofuran(300 mg) is added via addition funnel and the reaction is ambienttemperature for 1-2 hours. When the reaction is complete, the stirringis stopped, the layers are separated and the organic layer is washedtwice with water (2×6 g). The organic layer is dried over of sodiumsulfate (3 g), filtered and concentrated. Residual water is reducedfurther by concentration from fresh 2-methyltetrahydrofuran (6.5 g) andthe material is transferred as solution in anhydrous2-methyltetrahydrofuran to the next reaction.

Commercial 1 M K-Selectride in THF (1.20 g) is charged to a dry reactionvessel under a nitrogen atmosphere, diluted with anhydrous2-methyltetrahydrofuran (2.10 g) and cooled to −65° C. The solution of24f (0.41 g) in 2-methyltetrahydrofuran (1.5 g), is then slowly added tothe reaction vessel to control the internal temperature at −65±5° C. Thereaction is stirred for 2 hours and warmed to −20° C. over approximately1 hour and stirred for an additional hour. The reaction is monitored byHPLC and reactions that are incomplete are driven to completion withadditional K-selectride. The reaction is quenched at low temperaturewith MeOH (0.33 g), then 3M NaOH (2.4 g) at −20° C. and 15% hydrogenperoxide in water (1.04 g) at or below 5° C., then stirring overnight atambient temperatures. The layers are split and the organic layer iswashed sequentially with 1M aqueous NaOH (2 ml), 0.5 M aqueous Na₂SO₃ (2ml), and water (2 ml) adjusted to a pH of 3 with HCl. The organic layeris dried over sodium sulfate (0.82 g), filtered and concentrated. Theproduct 24 g (0.457 g) is re-concentrated from DCM (0.9 g) and used inthe next reaction.

24 g (1.36 g) is charged with anhydrous DCM (18.1 g) to an appropriatelysize reaction vessel, place under an inert atmosphere and cooled to −20°C. Triethylamine (0.61 mg) is charged followed by the slow addition ofmethanesulfonyl chloride (373 mg) in anhydrous DCM (300 mg). Thereaction is stirred for 1 hour at −20° C. The reaction is monitored byHPLC. Incomplete reactions are driven to completion with additionalmethanesulfonyl chloride. When complete, the reaction is quenched withwater (13.6 g) and allowed to warm. The layers are separated and theorganic layer is washed with 2.5 wt % sodium bicarbonate (13.8 g) andthen water (10.9 g). The organic layer is dried over of sodium sulfate(4 g), filtered, and concentrated. The product solution is solventexchanged via charge and concentration to t-butyl methyl ether (10.9 ml)and then 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU, 4.7ml). The DMPU solution is used directly in the next reaction.

Sodium azide (0.74 g) is charged to an appropriately sized reactionvessel. The solution of 24 h (1.46 g) in DMPU (5.9 g) is charged to thereaction vessel, rinsing with additional DMPU (1.9 g). The suspension isheated to 60° C. for 15 hours, maintaining a nitrogen sweep for theentire reaction. The reaction is cooled to ambient temperature anddiluted with MTBE (11.7 g). The organic solution is washed 3 times with2% saline (3×8 g), dried over sodium sulfate (4.4 g), filtered, andconcentrated. The product is concentrated from THF (6.4 g) and useddirectly in the next reaction.

The crude 24i (1.34 g) is dissolved and transferred to a suitably sizedreaction vessel with THF (12.6 g). Triphenylphosphine (0.70 g) and water(0.44 g) are charged and the reaction is heated to 55° C. for 15-24hours. When complete, the reaction is cooled to ambient temperature,dried with magnesium sulfate (1.4 g), filtered and concentrated. Thesolids are dissolved and concentrated from three portions of DCM (3×9 g)and purified by silica gel chromatography using DCM/MeOH/Et₃N gradientsto remove reagent based impurities. The pooled fractions areconcentrated to dryness, dissolved in DCM (6.8 g) and concentrated todryness again to afford an amorphous solid (1.12 g) which is used in thenext reaction.

24j (1.09 g) is dissolved and transferred to an appropriately sizedreaction vessel with anhydrous DCM (15.8 g) and placed under a nitrogenatmosphere. The solution is cooled to 0° C. N,N-diisopropylethylamine(357 mg) and neat methanesulfonyl chloride (0.165 ml) are chargedsequentially while maintaining temperature between below 5° C. Thereaction is monitored by HPLC. Incomplete reactions are driven tocompletion with additional methanesulfonyl chloride. The reaction isquenched with 0.4 M aqueous sodium bicarbonate (11.4 g) and warmed toambient temperature. The layers are separated and the aqueous phase isback extracted with DCM (5.8 g). The combined organic layers are driedover magnesium sulfate (0.55 g), filtered and concentrated. The product24k is dissolved and striped from 2-propanol (4.0 g) to remove residualDCM and used directly in the next reaction.

Aldrich Degussa type E101 NE/W 10% Pd/C (249 mg) is charged to anappropriately sized reaction vessel and placed under a nitrogenatmosphere. A 2-propanol (9.8 g) solution of 24k (1.24 g) is charged tothe reaction vessel. The system is degassed and placed under a nitrogenatmosphere, and the process is repeated with hydrogen. The reaction isstirred under a 1 atm of hydrogen at ambient temperature for 8 hours. Aninert atmosphere is returned to the vessel and a second charge ofcatalyst (125 mg) slurried in 2-propanol (0.5 g) is added to thereaction. The reaction mixture is degassed and placed under a nitrogenatmosphere, and the process is repeated with hydrogen. The reaction isstirred under 1 atm of hydrogen for another 15 hours at ambienttemperature. The reaction is monitored by HPLC. Incomplete reactions aretreated with additional catalyst and hydrogen. When complete, thereaction is filtered, treated with steam activated carbon (200 mg), andfiltered again. The solution is dried by partial concentrationtransferred to a reaction vessel and diluted with anhydrous 2-propanolto 0.09 M based on the theoretical yield. A 1.25 M HCl solution in2-propanol (1.64 g) is charged over 20 minutes. The hydrochloride saltcrystallizes slowly with gentle stirring and is isolated by filtration.The crystals are washed with 2-propanol (2.5 g) and vacuum dried toafford Compound 42 (916 mg, 80% yield) as a 1:1 IPA solvate.

Example 25

Step A

A round-bottom flask was charged with the amine 42 (1.1 g, 2.1 mmol, 1equiv.), dry tetrahydrofuran (10 ml) and pyridine (880 uL, 10.9 mmol, 5equiv.). The cooled (0° C.) mixture was treated with benzoylperoxide(1.6 g, 6.5 mmol, 3 equiv.). The mixture was stirred for 2 hours at 0°C. then overnight at 25° C. Reaction mixture diluted with MTBE andwashed with a mixture of saturated aqueous NaHCO₃ with 1 N NaOH untilthe layer split. The organic layer was collected and the aqueous wasre-extracted once with MTBE. Combined organic layers were washed withbrine, dry over Na₂SO₄, filtered and concentrated to dryness. The crudeoil was dissolved in 5 mL of CH₂Cl₂, loaded onto SiO₂ (40 g) column andeluted from hexanes/EtOAc (10% to 50%) to give the benzoyl derivative 48(380 mg) ([M+H]=625.4 m/z).

Step B

A round-bottom flask was charged with 48 (374 mg, 0.6 mmol, 1 equiv.)and MeOH (5 mL). The solution was treated at 25° C. in presence of 2 NKOH (0.3 mL, 0.6 mmol, 1 equiv.). The mixture was stirred for 3 h. Thesolvent was removed under vacuum. MTBE was added to the residue, whichwas neutralized with 1N HCl. The layers were cut and the aqueous layerwas extracted with two portions of CH₂Cl₂. Combined organic layers weredried over Na₂SO₄, filtered, and concentrated to dryness. The crudematerial (380 mg) was dissolved with CH₂Cl₂, loaded onto a SiO₂ column(12 g) and eluted with hexanes/EtOAc (0% to 100%) to give thehydroxylamine 47. The material was lyophilized from t-BuOH/7% H₂O togive 213 mg of 47 as a white powder ([M+H]=521.4 m/z).

Example 26

Step A

A heat-gun dried flask was charged with dry CH₂Cl₂ (5 mL) and benzylalcohol (785 uL, 7.58 mmol, 1.3 equiv.). The cooled (0° C.) solution wastreated with chlorosulfonyl isocyanate (506 uL, 5.83 mmol, 1 equiv.).Then, DMAP (1.4 g, 11.6 mmol, 2 equiv.) was added and the mixture wasstirred for 1 h at 25° C. After complete dissolution of DMAP, thereaction was clear for a short period. Then, a white fluffy precipitateformed. The mixture was diluted with CH₂Cl₂ (30 mL) and washed withthree portions (30 mL each) of water. The organic layer was dried overNa₂SO₄, filtered, and evaporated to dryness. The desired white solid 51was taken to the next step without purification.

Step B

A round-bottom flask was charged with 52 (30 mg, 0.053 mmol, 1 equiv.)and 51 (18 mg, 0.053 mmol, 1 equiv.). Both reagents were dissolved inCH₂Cl₂ (2 mL) and the solution was stirred at 25° C. The crude materialwas loaded onto a SiO₂ column (4 g) and eluted with hexanes/EtOAc (0% to50%) to give 16 mg of the sulfamoylated derivative 53 ([M+Na]=796.4m/z).

Step C

A round-bottom flask was charged with 53 (16 mg, 0.021 mmol, 1 equiv.)and 11 mg of 10% Pd/C (wet, Aldrich Degussa type E101). The material wassuspended in 2-propanol (3 mL). The flask was sealed and purged threetimes with hydrogen and left overnight under 1 atm of hydrogen. Theslurry was filtered through 0.2 micron Acrodisc, washed with 2-propanol,and the solvent was removed under vacuum. The residue was purificationby SiO₂ column (1 g) eluting with CH₂Cl₂/MeOH (5% to 10%). The majorproduct was lyophilized from t-BuOH/7% H₂O to give 9 mg of sulfamide 50([M+H]=506.4 m/z).

Example 27

Step A

A round-bottom flask was charged with cyclopamine 4-en-3-one (3.5 g, 8.5mmol, 1 equiv.) and pyridine (70 mL). The reactor was charged with Pd/C(10% Pd, 500 mg). The reaction was placed under 1 atmosphere ofhydrogen. After 3.5 hrs, LCMS showed complete consumption of startingmaterial. The catalyst was filtered off on an Acrodisk 0.2 micron filterand washed with toluene. The solvent was removed by azeotropic removalwith toluene (2×10 mL). The desired material 56, 3.5 g ([M+H]=412.5 m/z)was used as it for the next step.

Step B

A round-bottom flask was charged with 56 (1.2 g, 2.8 mmol, 1 equiv.),CH₂Cl₂ (10 mL) and triethylamine (1.9 mL, 14.2 mmol, 5 equiv.). Thecooled (0° C.) solution was treated with CBz-Cl (440 uL, 2.8 mmol, 1equiv.). After 1 hr, LCMS showed complete consumption of startingmaterial. The mixture was diluted with water. The layers were cut andthe organic layer was washed twice with water. The organic layer wasdried over sodium sulfate, filtered, and concentrated to dryness. Theproduct was purified by column chromatography (SiO2, 40 g) eluting withhexane/EtOAc (0 to 20%) to give 57 (891 mg) ([M+Na]=468.4 m/z).

Step C

In a round-bottom flask, the ketone 57 was azeotroped a couple timeswith CH₂Cl₂ and dried under vacuum for 1 h. Under nitrogen, the ketone 2(693 mg, 1.27 mmol, 1 equiv.) was dissolved in anhydrous THF (20 mL) andthe solution was cooled to −78 C. A 1 M solution of K-selectride in THF(1.9 mL, 1.9 mmol, 1.5 equiv.) was added dropwise. After 1 h, thereaction was complete by TLC. The reaction was quenched by addition of2.6 mL of 5 N NaOH followed by slow addition of 2.6 mL of 30% wt H₂O₂.The resulting mixture was allowed to stir overnight. The mixture waspartitioned between water and EtOAc. The aqueous layer was backextracted with EtOAc. The combined organic were washed first with water(buffered with a small portion of ammonium chloride) then with brine.The organic were dried, filtered, and concentrated to a crude foam (840mg) The crude material was dissolved in CH₂Cl₂, loaded on a SiO₂ column(40 g) and eluted with hexanes/EtOAc (0 to 50%) to give 58 (565 mg).

Step D

In a round-bottom flask under nitrogen, the alcohol 58 (530 mg, 0.98mmol, 1 equiv.) was dissolved in 5 mL of anhydrous CH₂Cl₂ andtriethylamine (800 uL, 5.81 mmol, 6 equiv.). The reaction mixture wascooled to 0° C. and Ms-Cl (112 uL, 1.45 mmol, 1.5 equiv) was addeddropwise. The mixture was stirred at 0° C. for 30 min. TLC(hexane:EtOAC, 7:3) showed ˜70% conversion. 70 uL of triethylamine (70uL, 0.5 equiv.) and Ms-Cl (10 uL, 0.1 equiv) were charged to thereaction vessel. After 90 min, a solution of saturated bicarbonate wascharged and the residue was extracted with CH₂Cl₂. The organic layer waswashed with water, dried and concentrated to a off-white foam. Thematerial was dissolved in CH₂Cl₂ and purified with SiO₂ (40 g) elutingwith hexanes/EtOAc (0% to 50%) to give 59 (430 mg).

Step E

In a round-bottom flask, the mesylate 59 (420 mg, 0.67 mmol, 1 equiv.)was dissolved in 2 mL of DMPU. The solution was treated with sodiumazide (218 mg, 3.4 mmol, 5 equiv.) at 60° C. for 5 h. The mixture wascooled to 25° C., then poured into ice-water to generate a white solid.The compound was extracted with MTBE (3 times). The combined organiclayers were washed with water (2×), then brine. The organic layers weredried over Na₂SO₄, filtered, and concentrated to a white foam (342 mg).The desired material 60 was used as is for the next step.

Step F

In a round-bottom flask equipped with a condenser, the azide 60 (336 mg,0.58 mmol, 1 equiv.) was dissolved in 7 mL of THF and 140 uL of waterand treated with triphenylphosphine (462 mg, 1.76 mmol, 3 equiv.). Themixture was heated to 70° C. overnight. TLC (hexane/EtOAc, 7:3)confirmed the reaction was complete. The reaction was concentrated todryness. The crude material was dissolved in CH₂Cl₂, loaded onto 12 g ofSiO₂ and eluted with CH₂Cl₂/MeOH (0 to 20%) to give the amine 61 (254mg).

Step G

In a round-bottom flask under nitrogen, the amine 61 (248 mg, 0.45 mmol,1 equiv.) was dissolved in 7 mL of anhydrous CH₂Cl₂ andN,N-diisopropylethylamine (237 uL, 0.91 mmol, 2 equiv.). The reactionmixture was cooled to 0° C. and Ms-Cl (70 uL, 1.45 mmol, 1.5 equiv) wasadded dropwise. The mixture was stirred at 0° C. for 2 h. TLC(hexane/EtOAc, 7:3) showed a little amount of amine. The mixture wascharged with 10 uL of Ms-Cl (0.2 equiv.), and warmed to 25° C. for 1 h.The reaction mixture was diluted with CH₂Cl₂ then a saturated solutionof NaHCO₃. The layers were cut. The aqueous layer was extracted with oneportion of CH₂Cl₂. The combined organic layers were washed with water,dried over Na₂SO₄, filtered and concentrated to dryness. The crude (326mg) was added to a SiO₂ column (12 g) and was eluted with hexanes/EtOAc(0 to 50%) to give the sulfonamide 62 (256 mg).

Step H

A round-bottom flask was charged with the sulfonamide 62 (250 mg, 0.4mmol, 1 equiv.) and 50 mg of 10% Pd/C (wet, Aldrich Degussa type E101lot 08331KC). The material was suspended in EtOAc (5 mL). The flask wassealed and purged three times with hydrogen and stirred under 1 atm ofhydrogen. After 3 h some conversion was observed, but a lot of startingmaterial remained. The slurry was filtered through 0.2 micron Acrodisc,washed with 2-propanol. The filtrate solution was re-subjected to thereaction condition by adding 54 mg of catalyst. The reaction wascompleted after 3 h. The slurry was filtered through 0.2 micronAcrodisc, washed with 2-propanol, and the solvent was concentrated todryness. The crude material (200 mg) was loaded onto a SiO₂ column (12g) and the compound was eluted using a gradient CH₂Cl₂/MeOH (0 to 10%)to give the free amine. The material was lyophilized from t-BuOH/7% H₂Oto give 175 mg of 55 as a white powder ([M+H]=491.3 m/z).

Example 28 Inhibition of the Hedgehog Pathway in Cell Culture

Hedgehog pathway specific cancer cell killing effects may be ascertainedusing the following assay. C3H10T1/2 cells differentiate intoosteoblasts when contacted with the sonic hedgehog peptide (Shh-N). Upondifferentiation; these osteoblasts produce high levels of alkalinephosphatase (AP) which can be measured in an enzymatic assay (Nakamuraet al., 1997 BBRC 237: 465). Compounds that block the differentiation ofC3H10T1/2 into osteoblasts (a Shh dependent event) can therefore beidentified by a reduction in AP production (van der Horst et al., 2003Bone 33: 899). The assay details are described below. The resultsapproximate (EC₅₀ for inhibition) of the differentiation assay is shownbelow in Table 1.

Assay Protocol

Cell Culture

Mouse embryonic mesoderm fibroblasts C3H10T1/2 cells (obtained fromATCC) were cultured in Basal MEM Media (Gibco/Invitrogen) supplementedwith 10% heat inactivated FBS (Hyclone), 50 units/ml penicillin and 50ug/ml streptomycin (Gibco/Invitrogen) at 37 C with 5% CO₂ in airatmosphere.

Alkaline Phosphatase Assay

C3H10T1/2 cells were plated in 96 wells with a density of 8×10³cells/well. Cells were grown to confluence (72 hrs). After sonicHedgehog (250 ng/ml), and/or compound treatment, the cells were lysed in110 μL, of lysis buffer (50 mM Tris pH 7.4, 0.1% TritonX100), plateswere sonicated and lysates spun through 0.2 μm PVDF plates (Corning). 40μL, of lysates was assayed for AP activity in alkaline buffer solution(Sigma) containing 1 mg/ml p-Nitrophenyl Phosphate. After incubating for30 min at 37° C., the plates were read on an Envision plate reader at405 nm. Total protein was quantified with a BCA protein assay kit fromPierce according to manufacturer's instructions. AP activity wasnormalized against total protein. Note that “A” indicates that the IC₅₀is less than 20 nM, “B” indicates that the IC₅₀ is 20-100 nM, “C”indicates that the IC₅₀ is >100 nM.

TABLE 1 Approximate EC₅₀ for Inhibition Compound Differentiation AssayEC₅₀ 1 A 7 C 8 C 9 C 10 C 13 A 20 A 21 B 22 A 23 A 24 A 27 B 29 B 31 B33 C 35 A 37 A 39 B 40 A 42 A 55 A

Example 29 Pancreatic Cancer Model

The activity of Compound 42 was tested in a human pancreatic model:BXPC-3 cells were implanted subcutaneously into the flanks of the rightlegs of mice. On day 42 post-tumor implant, the mice were randomizedinto two groups to receive either Vehicle (30% HPBCD) or Compound 42.Compound 42 was dosed at 40 mg/kg/day. After receiving 25 daily doses,Compound 42 statistically reduced tumor volume growth by 40% whencompared to the vehicle control (p=0.0309). At the end of the study, thetumors were harvested 4 hours post the last dose to evaluate an ontarget response by q-RT-PCR analysis of the HH pathway genes. Analysisof human Gli-1 resulted in no modulation. Analysis of murine Gli-1 mRNAlevels resulted in a robust down-regulation in the Compound treatedgroup, when compared to the Vehicle treated group.

Example 30 Medulloblastoma Model

The activity of Compound 42 was also evaluated in a transgenic mousemodel of medulloblastoma. Mice that are heterozygous for loss offunction mutations in the tumor suppressors Patched1 (Ptch1) andHypermethylated in Cancer (Hic1) develop spontaneous medulloblastoma.Similar to human medulloblastoma, these tumors demonstrate completepromoter hypermethylation of the remaining Hid 1 allele, as well as lossof expression of the wild type Ptch1 allele. When passaged assubcutaneous allografts, these tumors grow aggressively and are Hedgehogpathway-dependent. This model was employed to evaluate the efficacy oforally administered Compound, and to correlate activity with drugexposure in plasma and tumors. Oral administration (PO) of a single doseof Compound 42 led to dose-dependent down-regulation of the HH pathwayin subcutaneously implanted tumors, as measured by decreased Gli-1 mRNAexpression 8 hours post dose administration.

Daily (QD) administration of the Compound PO led to a dose dependentinhibition of tumor growth, with frank tumor regression seen at higherdoses. The approximate effective daily oral dose for 50% inhibition oftumor growth (ED50) is 4 mg/kg. When animals were treated QD for 21days, long term survival was observed following cessation of treatment(>60 days), with little to no tumor re-growth.

Example 31 Lung Cancer Model

To test the activity of Compound 42 in a human SCLC tumor model, LX22cells were implanted subcutaneously into the flank of the right leg.LX22 is primary xenograft model of SCLC derived from chemo-naivepatients, which has been maintained by mouse to mouse passaging. Thistumor responds to etoposide/carboplatin chemotherapy in way that closelyresembles a clinical setting. LX22 regresses during chemotherapytreatment, goes through a period of remission, and then begins to recur.In the LX22 model, Compound single agent activity and its ability tomodulate the chemoresistant recurrence was tested. On day 32 post tumorimplant, mice were randomized into three dosing groups to receiveVehicle (30% HBPCD), Compound, or the chemotherapy combination ofetoposide and carboplatin (E/P). Compound 42 was administered at a doseof 40 mg/kg/day, and after 16 consecutive doses there was no measurabledifference between the treated and vehicle groups. Etoposide wasadministered i.v at 12 mg/kg on days 34, 35, 36, and 48, whileCarboplatin was administered i.v. at 60 mg/kg on days 34, 41, and 48,post tumor implant. On day 50, the E/P treated mice were furtherrandomized to receive either Vehicle (30% HPBCD) or Compound follow uptreatment. The Compound was administered at the oral multi-dose MTD of40 mg/kg/day, and after 35 consecutive doses there was a substantialdelay in tumor recurrence in the treated group, compared to the vehiclegroup (p=0.0101).

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

It is claimed:
 1. A hydrochloric acid salt of a compound having thestructure:

wherein the hydrochloric acid salt is in the form of an iso-propanolsolvate.
 2. A pharmaceutical composition comprising the hydrochloricacid salt of claim 1 and a pharmaceutically acceptable excipient.